| Objective:Immunotherapy is the important part of tumor treatment. The main goal is to increase the specificity of the antitumor immune response and to limit the occurrence of autoimmune toxicity. Some clinical trials suggest that peptide-based vaccine have no serious side effects and have partial and complete tumor regression. In this research, mild acid wash method were used to collect the cytomembrane proteins in renal cell carcinoma 786-0 cell. Dendritic cells were obtained from peripheral blood mononuclear cells(PBMC). Then PBMC were cultivated with GM-CSF, IL-4 and autologous serum culture medium. Multiple antigen peptide DC cell vaccine was obtained by DC pulsed acid-eluted peptide. The tumor antigen specific CTL was generated from activated T cell by vaccine. ~51Cr-release assays to investigate the killing activity of the tumor antigen specific CTL activated by vaccine. Methods:1. Renal cell carcinoma 786-0 cells were cultivated in RPMI1640 culture medium supplemented with 10% fetal calf serum (FCS). RCC786-0were divided into two groups. One group was cultivated in culture medium added by hIFN- γ 2000u/ml, and another group no hIFN- γ .2. Analysis of the typing of HLA: The typing of HLA of RCC786-0 were performed by sequence specific primer polymerase chain reaction method, and the healthy adult volunteers were screened to match atleast one of the class I HLA of RCC786-0.3. Getting of multiple-antigen peptide: In this research mild acid wash method was used. Mild acid is made from citrate-phosphate bufferelution (0.131M citric acid and 0.066MNa2HPO4) .First collect bettergrowing 786-0 cells, then each flask cells were washed two times with phosphate-buffered saline(PBS) in order to remove culture medium. Anumber of 1 X 107 cells was treated with 5ml citrate-phosphate buffer elution at pH3.3 for lmin at room temperature. The eluent were centrifuged at 1000rpm X 5min,and stored at -70 ℃ .Then cells werewashed with PBS and culture medium for two times respectively, and them can be re-feeded with culture medium. After 24 hours they can be washed again by mild acid buffer elution. In the experiment one group of 786-0 cells were cultivated with culture medium supplemented with1000U/ml IFN. Then get HLA-1 associated peptides with the samemethod.4. The purification of acid eluted peptides: The eluent, which contain peptides inside, was desalted on Waters Sep-Pak C18 column accordingto the manufacturer's instructions, and concentrated by vacuum pump.Then acid eluted peptides were centrifuged on a Microcon YM-3(USA, Millipore) to get less than 3000MW peptides.5. The separation maturity of DC, and loaded with peptides to makevaccine: The volunteer's PBMC(peripheral blood mononuclear cells) were separated with density gradient centrifugation method. Then the PBMC were cultivated in RPMI1640 culture medium supplemented with GM-CSF lOOOU/ml, IL-4 500U/ml, 10% autologous serum and 37°C,5% CO2 condition. After 3 hours remove that are not dendritic cells.On 5th day, added acid eluted peptides of 6 X 107 cells to 1 X 104dendritic cells. The next day added LPS to maturate DC. At last, got two groups of dendritic cells vaccine with two groups of acid eluted peptides.6. The generation of tumor antigen specific CTL: The volunteer's PBMC were isolated. Then the PBMC were cultivated with hIL-2 and PHA after removing dendritic cells. After cultivated with hIL-2 for four weeks, the purified T lymphocytes were collected. Tumor antigen specific CTL was generated from activated T cell by vaccine.7. To investigate the killing activity of the tumor antigen specific CTL with 51Cr-release assay: With standard 4 hours 5ICr-release assay. Target cells were RCC786-0. Effector cells includes: One group of specific CTL specific cytotoxic T lymphocytes induced by multiple-antigen peptide DC cell accine which was got by R.CC786-0 cells cultivatedwith hlFN- Y ; Another group of specific CTL specific cytotoxic Tlymphocytes induced by multiple-antigen peptide DC cell vaccine; Lymphokine activated killer cells(LAK); T lymphocytes not activated by vaccine and T lymphocytes activated only by acid eluted peptides.Take the results less than zero, as zero. Show the results as X ± s, and check them with analysis of variance(ANOVA).51Cr release percentage^ (A—B) / (C—B) ] X100%A: experiment group cpm B: control group cpm C: maximal release control group cpm8. Analysis of acid eluted peptides with reverse-Phase High-performance Liquid Chromatography: Use SHIMADZU lOAvp HPLC machine. C18column(250x 4.6mm), SPD-lOAvp checking machine, wave 220nm; Solution A: 0.1 % TFA,99.9 % H2O; Solution B, 99.9 % acetonitrile, 0.1 % TFA. Flow rate is lml/min. Every time sample is 20 P L. Time program: 0—10min,100% A solution; 10—50min B solution 0—70%; 50—55min B solution 70%; 55—65mm B solution 70% — 100% ;65—70min 100 %B solution; 70—80min B solution 100%—0%.Results:1. The analysis of HLA: In the typing of HLA(human leukocyte antigen) in renal cell carcinoma 786-0 cells, A 03 is identified as main presentation. A healthy volunteer with heterozygote HLA-A03 was screened.2. Getting of multiple-antigen peptide: In acid condition(pH3.0—3.3),HLA-1 limited peptides can separate from cell surface together with P2 protein. In order to get enough antigen, we washed carcinoma cells for three times. After cultivated for 24 hours cells were washed againwith mild acid solution. Every time we washed about 2 X 107 number cells. hlFN- Y can improve the presentation of HLA-1 limited antige. In the research we got one group of cells activated by hlFN- y , then wegot two groups of acid eluted peptides.3. The purification of acid eluted peptides: The citrate phosphate and other salts were romoved away by Sep-pak Cl 8 column, then got rid of acetonitrile and water through concentrated by vacuum pump to getsolid peptides. Because HLA-1 limited peptides molecular weight areless than 3000dal, we can get that peptides by centrifuged on Microcon YM-3 after solid peptides dissolved in PBS.4. To investigate the killing activity of the tumor antigen specific CTL with 51Cr-release assay: With standard 4 hours 51Cr-release assay, investigate the immune response of multiple antigen peptide DC cell vaccine in renal cell carcinoma in vitro5. Analysis of acid eluted peptides with reverse-Phase High-performance Liquid Chromatography: In the research we find better time program:0~10min,100% A solution; 10~50min B solution 0—70% ; 50— 55min B solution 70%; 55—65min B solution 70 % — 100 % ; 65— 70min 100 %B solution; 70~80min B solution 100%~0%. Conclusions:This method is useful to get HLA- I limited peptides from RCC786-0cells by mild acid wash method. DC can recognize and present tumor antigens to CTL, then cause specific antitumor immune response. The results of the research shows that peptides got from cells induced by MFN-Y have not high immune response than another group peptides. Analysisof two groups peptides by reverse-Phase High-performance Liquid Chromatography suggests that every group has many single peptide distributing in different times peptide mount. It needs continue study aboutthat whether hlFN- Y can impact the presentation of peptide on tumor cellsurface.
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